WO2020170943A1 - Decellularization processing fluid and washing composition - Google Patents

Abstract

Provided is a decellularization processing fluid that allows efficient decellularization of a tissue without causing degradation of proteins that make up an extracellular matrix component. This decellularization processing fluid includes 0.01wt% to 20wt% of a sophorose lipid. The decellularization processing fluid may further include 0.1wt% to 10wt% of a surfactant. This decellularization processing fluid allows decellularization of a tissue without causing degradation of proteins that make up an extracellular matrix component.

Description

Decellularization treatment liquid and cleaning composition

The present invention relates to a tissue decellularization treatment solution.

In the field of transplantation medicine, when transplanting a graft derived from a living tissue of another person or another species, rejection of the graft by the tissue of the recipient is a problem. Therefore, in recent years, great expectations have been placed on the decellularized tissue. The decellularization technique is a technique in which cells causing a rejection reaction are removed from a biological tissue, and the remaining decellularized tissue, which is a supporting tissue, is used as it is or after being recellularized.

As a method of producing a decellularized tissue, a method of decellularizing a living tissue using a treatment solution containing a surfactant and a washing solution is known (see, for example, Patent Documents 1 to 4). However, in biological tissues that have been decellularized using a surfactant, the proteins that make up the extracellular matrix component (hereinafter extracellular matrix may be abbreviated as ECM) are degraded. And has low biocompatibility. Therefore, there are problems that the recellularization efficiency is poor and that thrombus is easily formed in the tissue.

In addition, the conventional method using a surfactant removes substances such as proteins and lipids by strongly adsorbing a surfactant having the property of forming micelles to the two-phase interface and significantly lowering the free energy of the interface. It was Such removal results in non-selective solubilization and washing out, which also affects extracellular matrix proteins, leaving the problem that tissue strength deteriorates and that surfactant remains in the tissue. It is difficult to control decellularization due to the effects on recellularization and transplantation. Furthermore, the use of a surfactant during washing has a problem of non-selectively removing decellularized extracellular matrix protein.

On the other hand, Sophorose lipids (hereinafter sometimes abbreviated as SL) have been discovered by PA Gorin et al. in a culture solution of Starmerella (Candida) bombicola (non-patented). Reference 1). Sophorolipid, which is known as one of biosurfactants, which is a surfactant of biological origin, is a fermentation product obtained from fermentation of yeast. Patent Document 5 describes the use of SL as a household cleaning agent having excellent foam stability. Patent Document 6 describes the use of SL as pharmaceuticals, cosmetics, and foods. However, these documents neither describe nor suggest the use of SL for decellularization treatment.

Japanese Patent Publication No. 2005-514971 Japanese Patent Publication No. 2006-507851 Special table 2005-531355 gazette JP, 2005-160040, A JP, 2017-145408, A Re-table 2015/137357

The present invention has been made in view of the above problems, and enables efficient decellularization of tissue while maintaining high mechanical strength without degrading the protein constituting the extracellular matrix component. It is intended to provide a decellularization treatment solution.

The decellularization treatment solution according to the present invention is a decellularization treatment solution used for decellularization of animal-derived tissues, and is characterized by containing 0.01% by weight or more and 20% by weight or less of sophorose lipid.

According to the present invention, a decellularization treatment liquid that enables efficient decellularization of tissue without degrading the protein that constitutes the extracellular matrix component can be obtained.

FIG. 3 is a photograph showing the effect of SL on decellularization of the lungs of newborn rats. FIG. 6 is a photographic diagram for confirming the decellularization effect of SL of newborn rat by SL compared with other drugs. It is a figure which shows the measurement result of the remaining amount of DNA after decellularization. It is a figure which shows the observation result by the confocal laser scanning microscope after decellularization. It is a figure which shows the measurement result of the area of the alveolar cavity after decellularization. It is a figure which shows the result of the immunostaining of collagen which is a main component of an extracellular matrix after decellularization. It is a figure which shows the observation result by the confocal laser scanning microscope after decellularization. It is a figure which shows the measurement result of the area of the alveolar cavity after decellularization. It is a figure which shows the result of the immunostaining of collagen which is a main component of an extracellular matrix after decellularization. It is a figure which shows the result of the immunostaining of collagen which is a main component of an extracellular matrix after decellularization. It is a figure which shows the measurement result of the area of the blood-vessel inner space after decellularization. It is a figure which shows the measurement result of the area of the blood-vessel inner space after decellularization. It is a photograph figure which confirms the decellularization effect of the liver of the rat newborn by SL. It is a photograph figure which confirms the decellularization effect of the rat newborn heart by SL. FIG. 6 is a photographic diagram for confirming the decellularization effect of SL on the neonatal rat kidney. FIG. 6 is a photographic diagram for confirming the decellularization effect on SL of newborn rat by SL in comparison with other drugs. It is a photograph figure which confirms the decellularization effect of the skin of the newborn rat by SL. It is a photograph figure which confirms the decellularization effect of the skin of the rat newborn by SL compared with other chemical|medical agents. FIG. 6 is a photograph showing the intestinal decellularization effect of newborn rats by SL. It is a photograph figure which confirms the decellularization effect of the spleen of the newborn rat by SL. FIG. 6 is a photographic diagram for confirming the decellularization effect on the neonatal rat spleen by SL in comparison with other drugs. FIG. 3 is a photographic diagram for confirming the decellularization effect on the heart of newborn rat and the shape maintenance effect of ECM by SL. FIG. 6 is a photographic diagram for confirming the effect of SL on ECM components after decellularization of the neonatal rat heart. FIG. 6 is a photographic diagram for confirming the change in volume after decellularization of the heart of a neonatal rat by SL. It is a photograph figure which confirms the decellularization effect to the heart of the newborn rat and the shape maintenance effect of ECM by SL compared with other agents.

Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings, the embodiments are for facilitating the understanding of the principle of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.

1. Decellularization treatment solution Since surfactants non-selectively remove substances such as proteins and lipids, the proteins that make up the extracellular matrix component are not present in the biological tissues that have been decellularized using detergents. It is deteriorated, leading to deterioration of strength of decellularized tissue and deterioration of cell re-adhesion performance, and low biocompatibility. Therefore, even when decellularization treatment is performed using sophorose lipid, which is one of the surfactants, the protein constituting the extracellular matrix component is degraded and biocompatibility is deteriorated, like other surfactants. Expected to be low. Contrary to this expectation, however, when the present inventor used sophorose lipid for the decellularization of animal-derived tissues, only the cells were removed and the decellularization of the tissues was carried out without degrading the protein constituting the extracellular matrix component. It was discovered as a new finding that the above is possible, and the present invention was completed based on this fact.

In the decellularization of the present invention, only the cells are selectively removed without affecting the extracellular matrix etc., but the mechanism can be considered as follows. Usually, the decellularization effect of a surfactant utilizes the action of removing cells from the extracellular matrix by the denaturing action of the membrane protein and the property of solubilizing the cell membrane and lysing the cell DNA. However, it is assumed that the extracellular matrix, which is a protein, also has a similar denaturing effect, and if it remains in the tissue, there is a concern of cytotoxicity. It is speculated that the decellularization of SL has the property of acting specifically only on lipids. Its action is that it acts only on the cell membrane and causes the cells to collapse. Therefore, it is considered that decellularization can be performed without affecting the extracellular matrix and its three-dimensional structure. Since SL is also low in cytotoxicity, it is considered that there is no concern about the use of organs and tissues after decellularization.

The decellularization treatment liquid according to the present embodiment is a decellularization treatment liquid used for decellularization of animal-derived tissues, and contains 0.01% by weight or more and 20% by weight or less of sophorose lipid.

Sophorose lipid is a glycolipid composed of sophorose or sophorose in which the hydroxyl group is partially acetylated, and hydroxyl fatty acid. In addition, sophorose is a sugar composed of two molecules of glucose with β1→2 bond. Hydroxyl fatty acid is a fatty acid having a hydroxyl group.

The sophorose lipid used in the following examples was prepared according to the description in JP-A-2016-160244.

Animal-derived tissues to be decellularized are not particularly limited, for example, pig, cow, horse, goat, sheep, rabbit, kangaroo, epithelial tissue obtained from mammals such as monkey and human, It is preferably at least one tissue selected from the group consisting of connective tissue, nerve tissue and muscle tissue, and specifically, heart, kidney, lung, liver, brain, intestine, uterus, omentum and small caliber blood vessel. Etc.

The decellularization treatment liquid according to the present embodiment contains 0.1% by weight or more and 20% by weight or less of sophorose lipid. This is because if the content of sophorose lipid is less than 0.1% by weight, the decellularization treatment may take time. On the other hand, when the content of sophorose lipid is more than 20% by weight, it is difficult to dissolve in the solution. The decellularization treatment solution according to the present embodiment is selected from 0.1% by weight, 1% by weight, 10% by weight, and 20% by weight of any two numerical values, and the sophorose lipid between the selected numerical values is selected. It can be included.

The decellularization treatment solution according to the present embodiment contains 0.1% by weight or more and 10% by weight or less of a surfactant in addition to 0.1% by weight or more and 20% by weight or less of sophorose lipid. The decellularization treatment of the conventional surfactant can be performed in a short time, and the influence of the conventional surfactant on the extracellular matrix can be reduced. This is because when the content of the surfactant is less than 0.1% by weight, the penetrating power into the tissue of animal origin may be poor, while when the content of the surfactant is more than 10% by weight. This is because there is a risk of damaging cells existing in animal-derived tissues.

The surfactant is not particularly limited, and examples thereof include an anionic surfactant, a cationic surfactant, an amphoteric surfactant, and a nonionic surfactant. Examples of the anionic surfactant include sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium α-olefinsulfonate, sodium lauryl phosphate, sodium laurate, triethanolamine laurate, sodium oleyl sarcosine, and sodium lauryl sarcosine. , Sodium palmityl sarcosine, sodium coconut oil fatty acid sarcosine, sodium oleyl glutamate, sodium lauryl glutamate, sodium palmityl glutamate, palm oil fatty acid glutamate triethanolamine, palm oil fatty acid glutamate sodium, lauroyl methyl-β-alanine sodium, ( Examples thereof include poly)oxyalkylene alkyl ether sulfate, (poly)oxyalkylene alkyl ether carboxylate, and (poly)oxyalkylene alkylsulfosuccinate. Examples of the cationic surfactant include stearyltrimethylammonium chloride, behenyltrimethylammonium chloride, distearyldimethylammonium chloride, lanolin fatty acid aminopropylethyldimethylammonium ethyl sulfate, diethylaminoethylamide stearate lactate, dilaurylamine hydrochloride, oleylamine. Examples include lactate. Examples of the amphoteric surfactant include lauryldimethylaminoacetic acid betaine, stearyldimethylaminoacetic acid betaine, coconut oil alkyldihydroxyethylaminoacetic acid betaine, lauryldihydroxyethylaminoacetic acid betaine, decyldihydroxypropylaminoacetic acid betaine, 2-alkyl-N-carboxymethyl -N-hydroxyethyl imidazolinium betaine, sodium octylaminopropionate, sodium laurylaminopropionate, coconut oil sodium alkylaminopropionate, sodium myristylaminopropionate, sodium palmitylaminopropionate, sodium stearylaminopropionate, lauryl Examples thereof include sodium aminoacetate and sodium laurylaminobutyrate, 2-[N,N-di(alkylbenzyl)-N-methylammonium]-ethylsulfate, N-stearyltaurine sodium and N-lauryltaurine sodium. As the nonionic surfactant, polyoxyethylene lauryl ether, glycerin monostearate, ethylene glycol monostearate, sorbitan monolaurate, methyl glucoside dioleate, polyoxyalkylene alkyl phenyl ether, polyoxyalkylene alkyl amino ether, poly Examples thereof include oxyethylene lauryl amine and lauryl dimethyl amine oxide.

Note that the decellularization treatment liquid according to the present embodiment can include a buffer, a chelating agent, an antiseptic, a bactericide, and an antioxidant.

Further, the decellularization treatment liquid according to the present embodiment can contain water, and examples of water include ultrapure water, ion-exchanged water, distilled water, tap water and industrial water.

2. Decellularized washing solution Recellularization of decellularized tissue occurs because the tissue residue and the substances such as the surfactant used for the decellularization process remain in the decellularized biological tissue. It is preferred to wash the remaining material before doing so.

The cleaning composition according to the present embodiment is a cleaning composition used for cleaning animal-derived tissue after decellularization treatment, and contains 0.01% by weight or more and 10% by weight or less of sophorose lipid, It is possible to remove only the residual material without damaging the outer matrix. This is because if the content of sophorose lipid is less than 0.01% by weight, the cleaning effect may not be sufficient, while if the content of sophorose lipid is more than 10% by weight, the tissue derived from animals may be This is because unnecessary sophorose lipid may remain. Preferably, the cleaning composition according to the present embodiment contains 0.1% by weight or more and 5% by weight or less of sophorose lipid, and more preferably 0.5% by weight or more and 1% by weight or less of sophorose lipid.

The cleaning composition according to the present embodiment further comprises at least one of acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, Tris buffer, HEPES buffer and MES buffer. It is possible to include a buffer solution containing

3. Method for Producing Decellularized Tissue One embodiment of the method for producing a decellularized tissue using the decellularization treatment solution according to the present embodiment will be described below.

First, wash the animal-derived tissue by immersing or perfusing the blood vessel in the animal-derived tissue with a buffer solution. The buffer solution used for washing is a buffer solution dissolved in water and is not particularly limited as long as it has a buffering action to keep the pH in the solution constant, but is preferably a phosphate buffer solution, More preferably, the solution contains an anticoagulant such as heparin. The pH of the buffer solution is preferably 4.0 to 9.0.

Next, the animal-derived tissue is immersed in a decellularization treatment solution containing 0.01% by weight or more and 20% by weight or less of sophorose lipid. The time for immersing the animal-derived tissue is not particularly limited as long as the decellularization effect is produced, but is, for example, 12 hours or more and 20 days or less, preferably 1 day or more and 15 days or less, and more preferably 2 days or more. 10 days or less. Instead of immersing the animal-derived tissue in the decellularization treatment liquid according to the present embodiment, it is also possible to decellularize the blood vessel in the animal-derived tissue by perfusing the decellularization treatment liquid.

Next, the animal-derived tissue after the decellularization step is washed, but if the decellularization treatment solution does not contain a surfactant other than sophorose lipid, or if it does not contain a surfactant, it is removed. It is possible to eliminate the need to wash the animal-derived tissue after the cell-forming step.

(Example 1) Confirmation of decellularization effect on SL of newborn rat by SL The newborn lung of rat was immersed in SL solution containing 10% by weight and 20% by weight of ultrapure water and shaken. After 1 day, 2 days, 5 days, and 10 days, the decellularization effect was confirmed using a stereoscopic microscope. The results are shown in Table 1 and FIG.

As shown in Table 1 and FIG. 1, when the rat neonatal lungs were immersed in 10 wt% or 20 wt% SL solution and shaken, the decellularization effect was good.

(Example 2) Confirmation of decellularization effect on SL of newborn rat by SL compared with other drugs 0.1 wt%, 1.0 wt%, 10 wt% SL solution, SDS (sodium lauryl sulfate) solution, Triton solution, The lungs of newborn rats were immersed in APG (alkyl polyglycoside) solution and shaken. After 2 days, 4 days, and 6 days, the decellularized state was evaluated by the transparency ratio using a stereomicroscope, and the effect on the extracellular matrix was visually reduced (size) or the collapsed state of the organ, tweezers. It was evaluated comprehensively by the strength when it was grasped with. As a more detailed evaluation of the effect on the extracellular matrix, the effect on the internal structure was confirmed by direct observation using a confocal laser microscope and immunostaining of collagen, which is a major component of the extracellular matrix. The evaluation results of the decellularized state are shown in Table 2-1, Table 2-2 and Table 2-3. The evaluation results of the effect on the extracellular matrix are shown in Table 3-1, Table 3-2 and Table 3-3. The results in Table 3 were confirmed by the very good days in Table 2. The photograph in FIG. 2 is a photograph 6 days after the immersion.

In addition, 0.01 wt% SL solution, 0.1 wt% SL solution, 0.5 wt% SL solution, 1.0 wt% SL solution, 0.1 wt% SDS solution, 1.0 wt% SDS solution, and mixed solution of SL solution + SDS solution (mixed solution The combination was shown in the table below), and the lungs of the newborn were soaked and shaken in the same manner as above. After 2 days, 4 days, and 6 days, the decellularized state was evaluated by the transparency ratio using a stereomicroscope, and the effect on the extracellular matrix was visually reduced (size) or the collapsed state of the organ, tweezers. It was evaluated comprehensively by the strength when it was grasped with. The evaluation results of the decellularized state are shown in Table 2-4. Table 3-4 shows the evaluation results of the effect on the extracellular matrix. The results in Table 3 were confirmed by the very good days in Table 2. As a more detailed evaluation of the effect on the extracellular matrix, the effect on the internal structure was confirmed by direct observation using a confocal laser scanning microscope and immunostaining of collagen, which is a major component of the extracellular matrix. In addition, the decellularized state was confirmed by measuring the amount of remaining DNA.

As shown in Table 2-4, it was confirmed that decellularization was completed in a short time by mixing sophorose lipid with a conventional surfactant.

In Example, redness due to cells disappeared after 4 days, and the decellularization effect was good. Since it was reported that the residual DNA amount was 50 ng/mg or less as a standard, it was confirmed that decellularization was good as shown in FIG.

Regarding the effect on the extracellular matrix, in the examples, there was no change in size, disintegration state, and strength, and no effect was observed. In the comparative example, an effect was observed in any of the items. This confirmed that the decellularized organs using SL had no effect on the extracellular matrix.

Also, when SL and SDS were mixed, no effect was observed on the extracellular matrix. In addition, it was confirmed that when SL and SDS were mixed, the effect on extracellular matrix decreased as the concentration of SL increased. In particular, when mixed with 1.0% by weight of SL, the effect on the extracellular matrix disappeared as in the case of SL alone.

Fig. 4 shows the results of confirmation of the internal structure of the lung by a confocal laser scanning microscope. Furthermore, the result of having measured the area of the alveolar cavity is shown in FIG. From FIGS. 4 and 5, a difference was found in the size of the alveolar cavity. It was confirmed that SL maintained the same size as the control, whereas SDS and Triton reduced the alveolar space. It was also confirmed that APG was smaller than SL. The results of observing collagen, which is a major constituent of the extracellular matrix, by immunostaining are shown in FIG. Compared with the control, SL treatment did not show a large change, whereas SDS and Triton showed remarkable shrinkage as a whole, and APG showed alveolar cavity collapse.

Fig. 7 shows the results of confirmation of the internal structure of the lungs by a confocal laser scanning microscope for the lungs that were decellularized with a mixture of SL solution and SDS solution. Furthermore, the result of having measured the area of the alveolar cavity is shown in FIG. From Figure 7 and Figure 8, treatment with 0.1% SDS or 1.0% SDS alone increases the alveolar cavity, but by adding 0.1% or more SL, the area of the alveolar cavity is similar to that of untreated control. It was confirmed that it is possible to suppress the expansion of the cyst. Based on these results, the shape-maintaining effect was confirmed by adding SL rather than SDS alone for decellularization of the lung.

Desirable decellularization effect is that only cells are removed and there is little effect on the extracellular matrix that is the skeleton of the organ. As effects on the extracellular matrix, morphological observation and extracellular matrix structure evaluation were performed. As a result, it can be confirmed that SL has no change in appearance and no influence on the internal structure during decellularization treatment as compared with other surfactants. It was found that only cells were selectively removed.

When the alveolar space shrinks, it affects cell invasion during recellularization, and it is possible that cells do not invade. In addition, when the extracellular matrix structure is broken, it is considered that the cells do not regenerate and adhere sufficiently even if they enter the cells, and thus cannot function as organs or tissues. Regarding the organs treated with SL, the size was maintained and the structure of the extracellular matrix was not disturbed and did not affect, so cell entry and cell adhesion during recellularization were better than treatment with other drugs. Yes, it can be said that it functions as the same function as the original organ.

∙ Regarding the organs other than the lungs, the appearance has not changed in SL processing. In addition, the major component of the extracellular matrix that composes each organ is collagen, and it is considered that the extracellular matrix structure is disrupted when treated with a substance other than SL, similar to the observation of the lung. It can be said that processing is superior.

(Example 3) Confirmation of the effect of SL on extracellular matrix of neonatal rat blood vessels After decellularization treatment with each treatment solution, primary antibody: Anti-Collagen IV, secondary antibody: Anti-Rabbit 488 It was used for immunostaining and observation. In addition, the lumen of the blood vessel was evaluated by measuring the area, and the effect on the extracellular matrix was confirmed. Observation photographs of immunostaining are shown in FIGS. 9 and 10, and area measurement results are shown in FIGS.

Regarding the blood vessels decellularized with 10% by weight of each solution, there was almost no change from the untreated blood vessels when SL was used, whereas the blood vessels treated with SDS and Triton had a reduced lumen, and It was confirmed that the blood vessels treated with APG had an expanded lumen. From this, it can be confirmed that the solutions other than SL have the effect of reducing or expanding the extracellular matrix of blood vessels. In addition, when treated with a mixed solution of SL solution and SDS solution, it was confirmed that the lumen of the blood vessel after decellularization approaches the lumen of the untreated blood vessel as the SL concentration becomes higher, and by adding SL SDS was also confirmed to have a shape-maintaining effect by suppressing the contraction of blood vessels that were contracted by itself.

(Example 4) Confirmation of decellularization effect of newborn rat liver by SL The newborn rat liver was immersed in SL solution containing 10% by weight and 20% by weight of ultrapure water and shaken. After 1 day, 2 days, 5 days, and 10 days, the decellularization effect was confirmed using a stereoscopic microscope. The results are shown in Table 4 and FIG.

As shown in Table 4 and FIG. 13, when the neonatal rat liver was immersed in 10% by weight or 20% by weight of SL solution and shaken, compared with the case where it was immersed in ultrapure water, the The cell effect was good.

(Example 5) Confirmation of decellularization effect on newborn rat heart by SL The newborn rat heart was immersed in SL solution of ultrapure water, 1% by weight, 10% by weight, and 20% by weight and shaken. .. After 1 day, 2 days, 5 days, and 10 days, the decellularization effect was confirmed using a stereoscopic microscope. The results are shown in Table 5 and FIG.

As shown in Table 5 and FIG. 14, when neonatal rat hearts were immersed in 1%, 10% or 20% by weight SL solution and shaken, By comparison, the decellularization effect was good.

(Example 6) Confirmation of decellularization effect of newborn rat kidney by SL The newborn rat kidney was immersed in ultrapure water, 10 wt%, 20 wt% SL solution and shaken. After 1 day, 2 days, 5 days, and 10 days, the decellularization effect was confirmed using a stereoscopic microscope. The results are shown in Table 6 and FIG.

As shown in Table 6 and FIG. 15, when the rat neonatal heart was immersed in 10 wt% or 20 wt% SL solution and shaken, the decellularization effect was good.

Next, newborn rat kidneys were immersed in 0.1 wt%, 1.0 wt%, and 10 wt% SL solution, SDS solution, Triton solution, and APG solution, and shaken. After 2 days, 4 days, 6 days, 8 days, and 10 days, the decellularized state was evaluated by the transparency ratio using a stereoscopic microscope, and the effect on the extracellular matrix was visually confirmed as the contracted state (size) of the organ, The state of organ collapse and the strength when grasped with tweezers were evaluated comprehensively. The results are shown in Tables 7 and 8. The results in Table 8 were confirmed by the very good days in Table 7. Decellularization was good. FIG. 16 shows a photograph 10 days after the immersion.

In the example, there was no change in size, collapsed state, and strength, and no effect was seen. The comparative example had an effect on either item. At all concentrations, there was no change in size, disintegration, or strength only in the decellularized sample using SL of Example.

(Example 7) Confirmation of decellularization effect of skin of newborn rat by SL Immersing skin of newborn rat in SL solution of ultrapure water, 0.1% by weight, 1% by weight, 10% by weight, 20% by weight , Shaken. After 1 day, 2 days, 5 days, and 10 days, the decellularization effect was confirmed using a stereoscopic microscope. The results are shown in Table 9 and FIG.

As shown in Table 9 and FIG. 17, when the neonatal rat skin was immersed in 0.1% by weight, 1% by weight, 10% by weight or 20% by weight of SL solution and shaken, it was treated with ultrapure water. The decellularization effect was better than that in the case of immersion.

The neonatal rat skin was immersed in 0.1% by weight, 1.0% by weight, and 10% by weight of SL solution, SDS solution, Triton solution, and APG solution, and shaken. After 2 days, 4 days, 6 days, 8 days, and 10 days, the transparency was evaluated by using a stereoscopic microscope. As the influence on the extracellular matrix, the contracted state (size) of the organ or the collapsed state of the organ, The strength when grasped with tweezers was comprehensively evaluated. The results are shown in Tables 10 and 11. The results in Table 11 were confirmed by the very good days in Table 10. FIG. 18 shows a photograph 10 days after the immersion. The results in Table 11 were confirmed by the very good days in Table 10. On the 4th day after the immersion, the redness of the cells was burnt out, and the decellularization was good.

Regarding the effect on the extracellular matrix, in Example, there was no change in size, disintegration state, and strength, and no effect was observed. The comparative example had an effect on either item. At all concentrations, there was no change in size, disintegration, or strength only in the decellularized sample using SL of Example.

(Example 8) Confirmation of decellularization effect on intestine of newborn rat by SL Immersion of intestine of newborn rat in SL solution of ultrapure water, 0.1% by weight, 1% by weight, 10% by weight, 20% by weight , Shaken. After 1 day, 2 days, 5 days, and 10 days, the decellularization effect was confirmed using a stereoscopic microscope. The results are shown in Table 12 and FIG.

As shown in Table 12 and FIG. 19, when the neonatal rat intestine was immersed in 0.1% by weight, 1% by weight, 10% by weight or 20% by weight of SL solution and shaken, it was treated with ultrapure water. The decellularization effect was better than that in the case of immersion.

(Example 9) Confirmation of decellularization effect on spleen of rat newborn by SL The spleen of newborn rat was immersed in SL solution of ultrapure water, 1% by weight, 10% by weight, and 20% by weight and shaken. .. After 1 day, 2 days, 5 days, and 10 days, the decellularization effect was confirmed using a stereoscopic microscope. The results are shown in Table 13 and FIG.

As shown in Table 13 and FIG. 20, when the intestine of a newborn rat was immersed in 1% by weight, 10% by weight or 20% by weight of SL solution and shaken, the decellularization effect was good. ..

Next, the spleen of a newborn rat was immersed in 0.1% by weight, 1.0% by weight, 10% by weight of SL solution, SDS solution, Triton solution, and APG solution, and shaken. After 2 days, 4 days, 6 days, 8 days, and 10 days, the decellularized state was evaluated by the transparency ratio using a stereoscopic microscope, and the effect on the extracellular matrix was visually confirmed as the contracted state (size) of the organ, The state of organ collapse and the strength when grasped with tweezers were evaluated comprehensively. The results are shown in Tables 14 and 15. The results in Table 15 were confirmed by the very good days in Table 14. FIG. 21 shows a photograph 10 days after the immersion.

Regarding the decellularization state, the redness disappeared on the 10th day, and decellularization became very good.

Regarding the effect on the extracellular matrix, in Example, there was no change in size, disintegration state, and strength, and no effect was observed. The comparative example had an effect on any of the items. At all concentrations, there was no change in size, disintegration, or strength only in the decellularized sample using SL of Example.

Example 10 Confirmation of Decellularization Effect and ECM (Extracellular Matrix) Shape-Maintaining Effect by Addition of SL to Newborn Rat Heart The newborn rat heart was removed and impregnated with the solution at room temperature and shaken for 5 days. went. Decellularization and shape maintenance were visually evaluated by confirming the transparency of the heart with transmitted light and a stereoscopic image with a stereoscopic microscope. The results are shown in Table 16 and FIG. In FIG. 22, the upper part of the same heart is a photographic image observed with a stereoscopic image, and the lower part is a photographic diagram observed with transmitted light.

As shown in Table 16 and FIG. 22, in the case of the decellularization treatment solution according to the present example, the decellularization effect was good, and the shape maintenance of ECM was also good.

(Example 11) Effect on ECM constituents after decellularization when SL was added to rat neonatal heart The rat neonatal heart was impregnated in the solution at 25°C (room temperature) for 24 hours and shaken, The existence and distribution of extracellular matrix constituent protein (fibronectin) were observed with a fluorescence stereomicroscope and a confocal microscope. The results are shown in Table 17 and FIG.

As shown in Table 17 and FIG. 23, in the case of the decellularization treatment liquid according to the present example, the ECM constituents were maintained well after decellularization.

(Example 12) Effect on recellularization after decellularization when SL was added to the heart of newborn rat The heart of newborn rat was impregnated with each solution at 25°C (room temperature) for 24 hours and shaken. The heart was washed for 6 hours. Then, neonatal rat cardiomyocytes (2×10 ⁶ ) were added and cultured from above. The pulsation of the heart 7 days after seeding was confirmed by morphological observation.

As shown in Table 18, in the case of the decellularization treatment liquid according to this example, recellularization after decellularization was successful.

(Example 13) Effect of SL addition on decellularized heart of rat newborn on washing The heart of newborn rat was impregnated with 1% SDS at 25°C (room temperature) for 24 hours and shaken. The composition was washed for 6 hours. Then, neonatal rat cardiomyocytes (2×10 ⁶ ) were added and cultured from above. The heart beat was confirmed 7 days after seeding.

As shown in Table 19, in the case of the decellularization treatment solution according to this example, recellularization after decellularization was successful.

(Example 14) Effect of SL addition on decellularized heart of rat newborn on washing: Newborn rat heart was impregnated with 1% SDS + 0.5% SL for 24 hours at 25°C (room temperature), and the heart was shaken. Was washed with the composition shown in Table 20 for 6 hours. Then, neonatal rat cardiomyocytes (2×10 ⁶ ) were added and cultured from above. The heart beat was confirmed 7 days after seeding.

(Example 15) Change in heart volume after decellularization when SL was added to rat newborn heart The rat newborn heart was impregnated and shaken at 25°C (room temperature) in the solution shown in Table 21 for 24 hours. After the treatment, the heart volume ratio was observed. The results are shown in Table 21 and FIG. In FIG. 24, the upper part of the same heart is a photographic image observed with a stereoscopic image, and the lower part is a photographic diagram observed with transmitted light.

It was found that the decellularization treatment solution according to the present example does not reduce the volume of the organs of animal cells.

(Example 16) Comparison between observation image after decellularization treatment by SL and observation image after decellularization treatment by SDS The heart of a neonatal rat was impregnated and shaken with 1.0% SL for 24 hours at 25°C (room temperature). After the treatment, it was observed with a fluorescence microscope. As shown in the upper part of FIG. 25, both collagen type 1 and type 4 retained the scaly structure similar to that of the living heart. In addition, α-actinin positive cells (cardiomyocytes) were not detected, and decellularization was confirmed.

The rat neonatal heart was impregnated with 1.0% SDS for 24 hours at 25°C (room temperature), shaken, and then observed with a fluorescence microscope. As shown in the lower part of FIG. 25, in the type 1 and type 4 collagen, which is a typical extracellular matrix of human heart, the three-dimensional structure of the protein was greatly deformed, and the gap was partially lost. As described above, it was confirmed that SL exhibits an excellent effect in retaining the biological structure in the decellularization step.

Can be used for tissue transplantation.

Claims (6)

1. A decellularization treatment liquid used for decellularization of animal-derived tissue, containing 0.01% by weight or more and 20% by weight or less of sophorose lipid.
2. The decellularization treatment liquid according to claim 1, further comprising a surfactant in an amount of 0.1% by weight or more and 10% by weight or less.
3. The decellularization treatment liquid according to claim 2, wherein the surfactant is sodium lauryl sulfate.
4. The decellularization treatment liquid according to claim 3, comprising 0.1% by weight or more and 1.0% by weight or less of sophorose lipid and 0.1% by weight or more and 1.0% by weight or less of sodium lauryl sulfate.
5. A cleaning composition used for cleaning animal-derived tissue after decellularization treatment, containing 0.01% by weight or more and 10% by weight or less of sophorose lipid.
6. The acetate buffer, the phosphate buffer, the citrate buffer, the borate buffer, the tartrate buffer, the Tris buffer, a buffer containing at least one of the HEPES buffer and the MES buffer according to claim 5. Cleaning composition.

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